An N-isopropylacrylamide polymer and a 2-(N,N-dimethylaminoethyl) methacrylate polymer are widely known as temperature-sensitive polymers exhibiting temperature sensitivity such that the polymers are hydrophilic at a lower temperature than the cloud point and hydrophobic at a temperature higher than the cloud point (Patent Literature 1 to 5).
As described in Patent Literature 1, poly(N-isopropylacrylamide) is hydrophobic near 37° C. and hydrophilic near 25° C., and these properties can be used to remove cultured cells from a culture dish. Cells adhere to a hydrophobic surface, and therefore when poly(N-isopropylacrylamide) is hydrophobic at 37° C., cells can be cultured on a culture dish to which poly(N-isopropylacrylamide) has been adhered. Upon the poly(N-isopropylacrylamide) becoming hydrophilic at 25° C., the cultured cells can be removed from the culture dish. Using this phenomenon, a cell sheet can be peeled off from the culture dish and collected.
In recent years, as the molecular genetic cause of human disease has become clear, more and more emphasis has been placed on gene therapy research. The purpose of gene therapy is to express DNA at the target site. What is crucial is how to cause the DNA to reach the target site, how to efficiently introduce the DNA into the target site, and how to express the DNA functionally at the site. As a vector for introducing foreign DNA, many viruses are modified to carry therapeutic genes and are used in clinical trials on humans for gene therapy. Such viruses include a retrovirus, adenovirus, adeno-associated virus, lentivirus, Sendai virus, and herpesvirus. The risk of infection or an immune response, however, still remains.
Patent Literature 3 discloses a vector with a branch structure, in which a branched chain extends radially with an aromatic ring such as benzene as the nucleus and discloses how this vector can condense DNA at a high density to form a small nucleic acid complex molecule for efficient gene transfer to a cell.
Patent Literature 2 discloses a gene transfer agent that uses the temperature sensitivity of poly[2-(N,N-dimethylaminoethyl) methacrylate]. A compound having three or more N,N-dialkyldithiocarbamylmethyl groups in the same molecule is used as an iniferter, and the gene transfer agent is composed of a star polymer formed by light irradiation living polymerization of 2-N,N-dimethylaminoethyl methacrylate and/or a derivative thereof with the iniferter. In this gene transfer agent, the 2-N,N-dimethylaminoethyl methacrylate and/or derivative thereof that forms the branched chain is slightly cationic. Therefore, the cationic 2-N,N-dimethylaminoethyl methacrylate unit contributes to bonding with the nucleic acid, increasing the amount of nucleic acid supported by the gene transfer agent. Furthermore, in this gene transfer agent, the branched chain is sensitive to temperature, and by increasing the temperature, the gene transfer agent becomes hydrophobic.
Patent Literature 5 discloses a gene transfer agent that can support more nucleic acid. A compound having three or more N,N-dialkyldithiocarbamylmethyl groups in the same molecule is used as an iniferter, and the gene transfer agent is formed by light irradiation living polymerization of 2-N,N-dimethylaminoethyl methacrylate and/or a derivative thereof with the iniferter.
In the gene transfer agent disclosed in Patent Literature 5, the 2-N,N-dimethylaminoethyl methacrylate and/or derivative thereof that forms the branched chain is slightly cationic. Therefore, the cationic 2-N,N-dimethylaminoethyl methacrylate unit contributes to bonding with the nucleic acid, increasing the amount of supported nucleic acid. Furthermore, since the branched chain is sensitive to temperature, the gene transfer agent can be made hydrophobic by adjusting the temperature.
For both of the polymers N-isopropylacrylamide and 2-(N,N-dimethylaminoethyl) methacrylate, the LCST (cloud point) is near 32° C. in an aqueous solution. Using temperature sensitive characteristics whereby these polymers are hydrophobic at the body temperature range of mammals and hydrophilic at room temperature, these polymers are used in the fields of drug delivery systems (DDS) and cell culturing. Due to the kinetic energy of polymer side chains and the affinity with water molecules of the side chain molecular group, these temperature sensitive characteristics manifest themselves by causing transfer between a globular structure and a coiled structure near 30° C.
The gene transfer agent disclosed in Patent Literature 3 has the properties of being hydrophilic at a temperature lower than a predetermined temperature (T) and hydrophobic at a temperature higher than the predetermined temperature (T). Patent Literature 4 discloses a culture vessel that, using these properties, has this gene transfer agent adhered to the inner surface of the vessel. In Patent Literature 4, the hydrophobic property of the gene transfer agent can be used for the gene transfer agent to adhere to the inner surface of the vessel over an extended period of time.
Reverse transfection is a technique used in gene transfer to adherent cells. The culture dish is coated in advance with nucleic acid complexes or the like, and genes are introduced from the side of the adhered base material using extended release of nucleic acid from the coating layer and incorporation by cells. This technique constitutes a pair with regular transfection, in which nucleic acid complexes are dispersed/dissolved in a culture medium and are incorporated into cells from the culture medium.